Reaction mixtures

ABSTRACT

The present invention provides stable dried reaction mixtures, methods for their preparation, methods for their use, and kits comprising them. The stable dried reaction mixtures are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).

CROSS-REFERENCE TO RELATED INVENTION

This application is a continuation of U.S. patent application Ser. No.14/958,645, filed Dec. 3, 2015, which claims the benefit of priority ofU.S. Provisional Patent Application Ser. No. 62/094,284, filed Dec. 19,2014, both of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention provides stable reaction mixtures, methods fortheir preparation, methods for their use, and kits comprising them. Thestable reaction mixtures are useful in many recombinant DNA techniques,especially nucleic acid amplification by the polymerase chain reaction(PCR).

BACKGROUND OF THE INVENTION

To address the challenge of producting stable polypeptide compositions,proteins are typically prepared in solid form to provide an acceptableshelf life. A standard method for preparing solid form proteincompositions is lyophilization (freeze-drying) but this process createsstresses which can denature proteins to varying degrees (Wang, W. (2000)International Journal of Pharmaceutics 203; 1-60). Few biologicalreaction compounds are stable in solubilized form for any length of timeand this is especially true for storage at room temperature.Consequently, numerous studies have been performed to evaluatepossibilities to enhance the storage capabilities of biological reactioncompounds in dry form. It is generally accepted that it will benecessary to use at least one stabilizing additive in order to assurethe biological activity of e.g. a polymerase upon re-solubilization.

WO 2008/36544 describes the use of so-called filler materials in orderto provide dried compositions, the filler materials are e.g.carbohydrates such as FICOLL, sucrose, glucose, trehalose, melezitose,DEXTRAN or mannitol, proteins such BSA, gelatin or collagen and polymerssuch as PEG or polyvinyl pyrrolidone (PVP). Glass-forming fillermaterials for stabilizing biological reagents are further described inU.S. Pat. Nos. 5,098,893, 5,200,399 and 5,240,843. The filler materialFICOLL is a copolymer disclosed in U.S. Pat. No. 3,300,474. The methodsof drying the liquid reaction mixtures are most of the time very complexin nature and therefore, the drying procedures are demanding andexpensive.

Freeze-drying (U.S. Pat. No. 5,593,824) or vacuum drying (U.S. Pat. No.5,565,318) is used for drying the biological materials in a carbohydratepolymer matrix. Lyophilization or freeze-drying is a well establishedtechnique towards storage of proteins that is disclosed in many state ofthe art documents (e.g. Passot, S., et al., Pharmaceutical Developmentand Technology 12 (2007) 543-553; Carpenter, J. F., et al.,Pharmaceutical Research 14 (8) (1997) 969-975; Schwegman, J. J., et al.,Pharmaceutical Development and Technology 10 (2005) 151-173).

A selection of drying conditions for different reaction mixtures forsequencing applications comprising genetic modifications of the Taqpolymerase are described in U.S. Pat. No. 7,407,747. Drying proceduresused are freeze-drying, speedvac without additional heat, speedvac withadditional heat and air drying at room temperature. The reactionmixtures within this patent were tested with respect to a variety ofcryoprotectants such as trehalose, sucrose, glucose andtrimethylamine-N-oxide (TMANO). Moreover, experiments were alsoperformed without cryoprotectants at all, but no data was disclosedconcerning the stability of those reaction mixtures with time. A goodstability for as long as 8 weeks was reported only for reaction mixturescomprising trehalose and bovine serum albumin (BSA).

Moreover, U.S. Pat. No. 7,407,747 discloses experiments with thepolymerase in different sequencing mixtures where each sequencingmixture comprises different compositions of buffer solution, nucleotidetriphosphates, and nucleotides with fluorescence label and primers.However, there is no disclosure if a polymerase in mixtures forreal-time PCR amplifications, namely mixtures comprising buffersolution, nucleotides triphosphates, primers and detection probes, maybe dried and stored without affecting the PCR activity of thepolymerase.

U.S. Pat. No. 8,652,811 discloses a method to dry a Taq DNA polymerasewithin a real-time PCR mixture, whereas the obtained dry composition canbe stored without affecting the PCR performance of the Taq DNApolymerase.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for stable reaction mixtures used fornucleic acid amplification by PCR and RT-PCR (Reverse Transcriptase-PCR)that comprises a saccharide (for example, sucrose) which have been drieddown without the need for lyophilization or freeze-drying. The presentinvention also provides for methods for preparing dried-down reactionmixtures.

Therefore in one aspect, the present invention involves a dry reactionmixture composition comprising at least one nucleic acidamplification-related enzyme, nucleoside monomers, and a saccharide,wherein the composition is non-lyophilized. In another aspect, thepresent invention involves a method of preparing a dry reaction mixturecomposition, the method comprising drying a reaction mixture in aqueousform in the absence of lyophilization, wherein the reaction mixture inaqueous form comprises at least one nucleic acid amplification-relatedenzyme, nucleoside monomers, and a saccharide. The embodiments andadvantages of the invention are described in more detail in the DetailedDescription of the Invention and in the Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the RT-PCR growth curves generated by the lyophilized (A)and non-lyophilized (B) dry reaction mastermixes on day 1 of storage at42° C.

FIG. 2 shows the RT-PCR growth curves generated by the lyophilized (A)and non-lyophilized (B) dry reaction mastermixes on day 15 of storage at42° C.

FIG. 3 shows the RT-PCR growth curves generated by the lyophilized (A)and non-lyophilized (B) dry reaction mastermixes on day 29 of storage at42° C.

FIG. 4 shows the RT-PCR growth curves generated by the lyophilized (A)and non-lyophilized (B) dry reaction mastermixes on day 49 of storage at42° C.

FIG. 5 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 90 of storage at 42° C.

FIG. 6 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 26 of storage at ambient temperature.

FIG. 7 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 71 of storage at ambient temperature.

FIG. 8 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 222 of storage at ambient temperature.

FIG. 9 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 299 of storage at ambient temperature.

FIG. 10 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 353 of storage at ambient temperature.

FIG. 11 shows the RT-PCR growth curves generated by the non-lyophilizeddry reaction mastermixes on day 379 of storage at ambient temperature.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides stable reaction mixtures, methods fortheir preparation, methods for their use, and kits comprising them. Thestable reaction mixtures are useful in many recombinant DNA techniques,especially nucleic acid amplification by the polymerase chain reaction(PCR). In particular, the present invention provides stablenon-lyophilized reaction mixtures containing an enzyme (e.g., an enzymeused in nucleic acid amplification) and a sugar. Such enzyme mixturesmay be dried down in the absence of lyophilization and stored long termat room temperature without significant loss of enzyme activity.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although essentially anymethods and materials similar to those described herein can be used inthe practice or testing of the present invention, only exemplary methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise.

The term “lyophilization” refers to the creation of a stable preparationof a biological substance by rapid freezing and dehydration of thefrozen product under high vacuum and is also commonly referred as“freeze-drying”.

The term “non-lyophilized”, “dried down” or “dried” refers to a processfor drying down a biological substance by not utilizing the process oflyophilization.

The term “ambient temperature” refers to the temperature of thesurrounding and is synonymous with “room temperature” when referring tothe temperature of a temperature-controlled indoor building. Typically,ambient temperature refers to a temperature range of between 15° C. and25° C. although slightly cooler or warmer temperatures may still beconsidered within the range of ambient temperature.

The term “aptamer” refers to a single-stranded DNA that recognizes andbinds to DNA polymerase, and efficiently inhibits the polymeraseactivity as described in U.S. Pat. No. 5,693,502, hereby expresslyincorporated by reference herein in its entirety. Use of aptamer anddUTP/UNG in RT-PCR is also discussed, for example, in Smith, E. S. etal, (Amplification of RNA: High-temperature Reverse Transcription andDNA Amplification with a Magnesium-activated Thermostable DNAPolymerase, in PCR Primer: A Laboratory Manual, 2nd Edition,Dieffenbach, C. W. and Dveksler, G. S., Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 211-219, (2003)).

“Recombinant”, as used herein, refers to an amino acid sequence or anucleotide sequence that has been intentionally modified by recombinantmethods. By the term “recombinant nucleic acid” herein is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid by restriction endonucleases, in a formnot normally found in nature. Thus an isolated, mutant DNA polymerasenucleic acid, in a linear form, or an expression vector formed in vitroby ligating DNA molecules that are not normally joined, are bothconsidered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell, it will replicate non-recombinantly, i.e., using thein vivo cellular machinery of the host cell rather than in vitromanipulations; however, such nucleic acids, once produced recombinantly,although subsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation.

The term “host cell” refers to both single-cellular prokaryote andeukaryote organisms (e.g., bacteria, yeast, and actinomycetes) andsingle cells from higher order plants or animals when being grown incell culture.

The term “vector” refers to a piece of DNA, typically double-stranded,which may have inserted into it a piece of foreign DNA. The vector ormay be, for example, of plasmid origin. Vectors contain “replicon”polynucleotide sequences that facilitate the autonomous replication ofthe vector in a host cell. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell, which, for example,replicates the vector molecule, encodes a selectable or screenablemarker, or encodes a transgene. The vector is used to transport theforeign or heterologous DNA into a suitable host cell. Once in the hostcell, the vector can replicate independently of or coincidental with thehost chromosomal DNA, and several copies of the vector and its insertedDNA can be generated. In addition, the vector can also contain thenecessary elements that permit transcription of the inserted DNA into anmRNA molecule or otherwise cause replication of the inserted DNA intomultiple copies of RNA. Some expression vectors additionally containsequence elements adjacent to the inserted DNA that increase thehalf-life of the expressed mRNA and/or allow translation of the mRNAinto a protein molecule. Many molecules of mRNA and polypeptide encodedby the inserted DNA can thus be rapidly synthesized.

The term “nucleotide,” in addition to referring to the naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, shall hereinbe understood to refer to related structural variants thereof, includingderivatives and analogs, that are functionally equivalent with respectto the particular context in which the nucleotide is being used (e.g.,hybridization to a complementary base), unless the context clearlyindicates otherwise.

The term “nucleic acid” or “polynucleotide” refers to a polymer that canbe corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleicacid (DNA) polymer, or an analog thereof. This includes polymers ofnucleotides such as RNA and DNA, as well as synthetic forms, modified(e.g., chemically or biochemically modified) forms thereof, and mixedpolymers (e.g., including both RNA and DNA subunits). Exemplarymodifications include methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, and the like), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen,and the like), chelators, alkylators, and modified linkages (e.g., alphaanomeric nucleic acids and the like). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Typically, the nucleotide monomers are linked viaphosphodiester bonds, although synthetic forms of nucleic acids cancomprise other linkages (e.g., peptide nucleic acids as described inNielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be orcan include, e.g., a chromosome or chromosomal segment, a vector (e.g.,an expression vector), an expression cassette, a naked DNA or RNApolymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, and a primer. A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence optionally comprises or encodes complementarysequences, in addition to any sequence explicitly indicated.

The term “oligonucleotide” refers to a nucleic acid that includes atleast two nucleic acid monomer units (e.g., nucleotides). Anoligonucleotide typically includes from about six to about 175 nucleicacid monomer units, more typically from about eight to about 100 nucleicacid monomer units, and still more typically from about 10 to about 50nucleic acid monomer units (e.g., about 15, about 20, about 25, about30, about 35, or more nucleic acid monomer units). The exact size of anoligonucleotide will depend on many factors, including the ultimatefunction or use of the oligonucleotide. Oligonucleotides are optionallyprepared by any suitable method, including, but not limited to,isolation of an existing or natural sequence, DNA replication oramplification, reverse transcription, cloning and restriction digestionof appropriate sequences, or direct chemical synthesis by a method suchas the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99,1979); the phosphodiester method of Brown et al. (Meth. Enzymol.68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al.(Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucciet al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesismethods; or the solid support method of Caruthers et al. U.S. Pat. No.4,458,066, or other methods known to those skilled in the art. All ofthese references are incorporated by reference.

The term “primer” as used herein refers to a polynucleotide capable ofacting as a point of initiation of template-directed nucleic acidsynthesis when placed under conditions in which polynucleotide extensionis initiated (e.g., under conditions comprising the presence ofrequisite nucleoside triphosphates (as dictated by the template that iscopied) and a polymerase in an appropriate buffer and at a suitabletemperature or cycle(s) of temperatures (e.g., as in a polymerase chainreaction)). To further illustrate, primers can also be used in a varietyof other oligonuceotide-mediated synthesis processes, including asinitiators of de novo RNA synthesis and in vitro transcription-relatedprocesses (e.g., nucleic acid sequence-based amplification (NASBA),transcription mediated amplification (TMA), etc.). A primer is typicallya single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). Theappropriate length of a primer depends on the intended use of the primerbut typically ranges from 6 to 40 nucleotides, more typically from 15 to35 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with a template forprimer elongation to occur. In certain embodiments, the term “primerpair” means a set of primers including a 5′ sense primer (sometimescalled “forward”) that hybridizes with the complement of the 5′ end ofthe nucleic acid sequence to be amplified and a 3′ antisense primer(sometimes called “reverse”) that hybridizes with the 3′ end of thesequence to be amplified (e.g., if the target sequence is expressed asRNA or is an RNA). A primer can be labeled, if desired, by incorporatinga label detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. For example, useful labels include³²P, fluorescent dyes, electron-dense reagents, enzymes (as commonlyused in ELISA assays), biotin, or haptens and proteins for whichantisera or monoclonal antibodies are available.

The term “conventional” or “natural” when referring to nucleic acidbases, nucleoside triphosphates, or nucleotides refers to those whichoccur naturally in the polynucleotide being described (i.e., for DNAthese are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATPcan be utilized in place of dATP in in vitro DNA synthesis reactions,such as sequencing. Collectively, these may be referred to as dNTPs.

The term “unconventional” or “modified” when referring to a nucleic acidbase, nucleoside, or nucleotide includes modification, derivations, oranalogues of conventional bases, nucleosides, or nucleotides thatnaturally occur in a particular polynucleotide. Certain unconventionalnucleotides are modified at the 2′ position of the ribose sugar incomparison to conventional dNTPs. Thus, although for RNA the naturallyoccurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP,collectively rNTPs), because these nucleotides have a hydroxyl group atthe 2′ position of the sugar, which, by comparison is absent in dNTPs,as used herein, ribonucleotides are unconventional nucleotides assubstrates for DNA polymerases. As used herein, unconventionalnucleotides include, but are not limited to, compounds used asterminators for nucleic acid sequencing. Exemplary terminator compoundsinclude but are not limited to those compounds that have a 2′,3′ dideoxystructure and are referred to as dideoxynucleoside triphosphates. Thedideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP arereferred to collectively as ddNTPs. Additional examples of terminatorcompounds include 2′-PO₄ analogs of ribonucleotides (see, e.g., U.S.Application Publication Nos. 2005/0037991 and 2005/0037398, which areboth incorporated by reference). Other unconventional nucleotidesinclude phosphorothioate dNTPs ([α-S]dNTPs), 5′-[α-borano]-dNTPs,[α]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs).Unconventional bases may be labeled with radioactive isotopes such as³²P, ³³P, or ³⁵S; fluorescent labels; chemiluminescent labels;bioluminescent labels; hapten labels such as biotin; or enzyme labelssuch as streptavidin or avidin. Fluorescent labels may include dyes thatare negatively charged, such as dyes of the fluorescein family, or dyesthat are neutral in charge, such as dyes of the rhodamine family, ordyes that are positively charged, such as dyes of the cyanine family.Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NANand ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G,and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are marketed byPerkin-Elmer (Boston, Mass.), Applied Biosystems (Foster City, Calif.),or Invitrogen/Molecular Probes (Eugene, Oreg.). Dyes of the cyaninefamily include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE HealthcareUK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

The term “Cp value” or “crossing point” value refers to a value thatallows quantification of input target nucleic acids. The Cp value can bedetermined according to the second-derivative maximum method (VanLuu-The, et al., “Improved real-time RT-PCR method for high-throughputmeasurements using second derivative calculation and double correction,”BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In thesecond derivative method, a Cp corresponds to the first peak of a secondderivative curve. This peak corresponds to the beginning of a log-linearphase. The second derivative method calculates a second derivative valueof the real-time fluorescence intensity curve, and only one value isobtained. The original Cp method is based on a locally defined,differentiable approximation of the intensity values, e.g., by apolynomial function. Then the third derivative is computed. The Cp valueis the smallest root of the third derivative. The Cp can also bedetermined using the fit point method, in which the Cp is determined bythe intersection of a parallel to the threshold line in the log-linearregion (Van Luu-The, et al., BioTechniques, Vol. 38, No. 2, February2005, pp. 287-293). The Cp value provided by the LightCycler instrumentoffered by Roche by calculation according to the second-derivativemaximum method.

The term “PCR efficiency” refers to an indication of cycle to cycleamplification efficiency. PCR efficiency is calculated for eachcondition using the equation: % PCR efficiency=(10^((−slope))−1)×100,wherein the slope was calculated by linear regression with the log copynumber plotted on the y-axis and Cp plotted on the x-axis. PCRefficiency can be measured using a perfectly matched or mismatchedprimer template.

The term “FRET” or “fluorescent resonance energy transfer” or “Foersterresonance energy transfer” refers to a transfer of energy between atleast two chromophores, a donor chromophore and an acceptor chromophore(referred to as a quencher). The donor typically transfers the energy tothe acceptor when the donor is excited by light radiation with asuitable wavelength. The acceptor typically re-emits the transferredenergy in the form of light radiation with a different wavelength. Whenthe acceptor is a “dark” quencher, it dissipates the transferred energyin a form other than light. Whether a particular fluorophore acts as adonor or an acceptor depends on the properties of the other member ofthe FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRApair. Commonly used quenchers are DABCYL and TAMRA. Commonly used darkquenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies,Inc., Novato, Calif.), Iowa Black™ (Integrated DNA Tech., Inc.,Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry &Assoc., Dexter, Mich.).

The terms “cell”, “cell line”, and “cell culture” can be usedinterchangeably and all such designations include progeny. Thus, thewords “transformants” or “transformed cells” include the primarytransformed cell and cultures derived from that cell without regard tothe number of transfers. All progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same functionality as screened for in the originallytransformed cell are included in the definition of transformants. Thecells can be prokaryotic or eukaryotic.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for procaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, positive retroregulatory elements (see U.S. Pat. No.4,666,848, incorporated herein by reference), and possibly othersequences. Eucaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

The term “operably linked” refers to the positioning of the codingsequence such that control sequences will function to drive expressionof the protein encoded by the coding sequence. Thus, a coding sequence“operably linked” to control sequences refers to a configuration whereinthe coding sequences can be expressed under the direction of a controlsequence.

The terms “restriction endonucleases” and “restriction enzymes” refer toenzymes, typically bacterial in origin, which cut double-stranded DNA ator near a specific nucleotide sequence.

Families of amino acid residues having similar side chains are definedherein. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., asparagine,glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, cysteine, glycine), beta-branched side chains(e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan, histidine).

The term “reagent solution” is any solution containing at least onereagent needed or used for PCR purposes. Most typical ingredients arepolymerase, nucleotide, primer, ions, magnesium, salts, pH bufferingagents, nucleotide triphosphates (NTPs) or deoxynucleotide triphosphates(dNTPs), probe, fluorescent dye (may be attached to probe), nucleic acidbinding agent, a nucleic acid template. The reagent may also be otherpolymerase reaction additive, which has an influence on the polymerasereaction or its monitoring.

The term “mastermix” refers to a mixture of all or most of theingredients or factors necessary for PCR to occur, and in some cases,all except for the template and primers which are sample and ampliconspecific. Commercially available mastermixes are usually concentratedsolutions. A mastermix may contain all the reagents common to multiplesamples, but it may also be constructed for one sample only. Usingmastermixes helps to reduce pipetting errors and variations betweensamples due to differences between pipetted volumes.

The term “thermostable polymerase” refers to an enzyme that is stable toheat, is heat resistant and retains sufficient activity to effectsubsequent primer extension reactions after being subjected to theelevated temperatures for the time necessary to denature double-strandednucleic acids. Heating conditions necessary for nucleic aciddenaturation are well known in the art and are exemplified in U.S. Pat.Nos. 4,965,188 and 4,889,818, which are incorporated herein byreference. As used herein, a thermostable polymerase is suitable for usein a temperature cycling reaction such as PCR. The examples ofthermostable nucleic acid polymerases include Thermus aquaticus Taq DNApolymerase, Thermus sp. Z05 polymerase, Thermus flavus polymerase,Thermotoga maritima polymerases, such as TMA-25 and TMA-30 polymerases,Tth DNA polymerase, and the like.

A “modified” thermostable polymerase refers to a polymerase in which atleast one monomer differs from the reference sequence, such as a nativeor wild-type form of the polymerase or another modified form of thepolymerase. Exemplary modifications include monomer insertions,deletions, and substitutions. Modified polymerases also include chimericpolymerases that have identifiable component sequences (e.g., structuralor functional domains, etc.) derived from two or more parents. Alsoincluded within the definition of modified polymerases are thosecomprising chemical modifications of the reference sequence. Theexamples of modified thermostable polymerases include G46E E678G CS5 DNApolymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671FCS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, aG46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase,ΔZ05-Gold polymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678GTMA-25 polymerase, E678G TMA-30 polymerase, and the like.

The term “thermoactive polymerase” refers to an enzyme that is active atthe elevated temperatures necessary to ensure specific priming andprimer extension (e.g., 55-80° C.).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably. The terms “nucleic acid” and “polynucleotide” are usedinterchangeably. Amino acid sequences are written from amino terminus tocarboxy terminus, unless otherwise indicated. Single-stranded nucleicacid sequences are written 5′ to 3′, unless otherwise indicated. The topstrand of a double-stranded nucleic acid sequence is written 5′ to 3′,and the bottom strand is written 3′ to 5′, unless otherwise indicated.

II. Dry Reaction Mixture Compositions

The dry reaction mixture composition of the present invention comprisesof at least one nucleic acid amplification-related enzyme, nucleotidetriphosphates, and a saccharide, wherein the composition isnon-lyophilized. The dry reaction mixture of the present invention hasimproved stability compared to a dry reaction mixture that lacks asaccharide or that is lyophilized, or that both lacks a saccharide andis lyophilized. In one embodiment of the dry reaction mixture, thesaccharide is chosen from sucrose, trehalose, dextrose, lactose,maltose, trehalose, cyclodextrins, maltodextrins and dextrans. Inanother embodiment, the dry reaction mixture retains activity uponstorage under conditions that are, or are equivalent to, 45° C. for 3months. In another embodiment, the dry reaction mixture retains activityupon storage under conditions that are, or are equivalent to ambienttemperature for 12 months. In yet another embodiment, the dry reactionmixture has at least one component selected from the group consisting ofan aptamer, a detergent, a buffer, a salt and an oligonucleotide. In yetanother embodiment, the dry reaction mixture further comprises manganeseacetate (Mn(OAc)₂). In still another embodiment, the dry reactionmixture has an amplification-related enzyme that is a thermostablepolymerase that is selected from the group consisting of Thermusaquaticus Taq DNA polymerase, Thermus sp. Z05 polymerase, Thermus flavuspolymerase, Thermotoga maritima polymerases, such as TMA-25 and TMA-30polymerases, Tth DNA polymerase, as well as modified thermostablepolymerases, or any combination thereof. In still another embodiment,the dry reaction mixture composition has an saccharide that is presentin an amount such that if the composition is reconstituted in aqueoussolution, the concentration of the saccharide is between about 50 mM toabout 1000 mM. In one further embodiment, the saccharide is sucrose.

III. Method of Preparing Dry Reaction Mixture Compositions

The dry reaction mixture composition of the present invention isprepared by drying the reaction mixture in aqueous form in the absenceof lyophilization, wherein the reaction mixture in aqueous formcomprises at least one nucleic acid amplification-related enzyme,nucleotide triphosphates, and a saccharide. In one embodiment, thedrying is performed at ambient temperature. In another embodiment, thesaccharide in the dry reaction mixture is at a concentration betweenabout 50 mM and about 1000 mM in the reaction mixture in aqueous form.In one further embodiment, the saccharide is sucrose. In yet anotherembodiment, the dry reaction mixture further comprises manganese acetate(Mn(OAc)₂). In still another embodiment, the dry reaction mixture has anamplification-related enzyme that is a thermostable polymerase that isselected from the group consisting of Thermus aquaticus Taq DNApolymerase, Thermus sp. Z05 polymerase, Thermus flavus polymerase,Thermotoga maritima polymerases, such as TMA-25 and TMA-30 polymerases,Tth DNA polymerase, as well as modified thermostable polymerases, or anycombination thereof. In yet another embodiment, the dry reaction mixturecomposition prepared using the method of the present invention retainsactivity upon storage under conditions that are, or are equivalent to,45° C. for 3 months. In yet another embodiment, the dry reaction mixturecomposition prepared using the method of the present invention retainsactivity upon storage under conditions that are, or are equivalent to,ambient temperature for 12 months.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the compositions and methods described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

The following examples are given to illustrate embodiments of thepresent invention as it is presently preferred to practice. It will beunderstood that the examples are illustrative, and that the invention isnot be considered as restricted except as indicated in the appendedclaims.

EXAMPLES Example 1: Preparation of Dry Reaction 5×RT-PCR Mastermixes

Dry Reaction 5×RT-PCR mastermixes were first prepared from aqueoussolutions with the following composition: 250 mM Tricine (pH 8.3), 500mM KOAc (pH 7.5), 0.05% Tween 20, 1.326 μM Aptamer (used for hot-startPCR), 1000 μm dATP, 1000 μm dCTP, 1000 μm dGTP, 1500 μm dUTP, 150 μmdTTP, 0.029% NaN₃, 0.5 mM EDTA, 0.2 U/μl uracil-N-glycosylase (UNG), 2.2U/μl Z05-D DNA polymerase. 1.5M sucrose was added in half of the5×RT-PCR mastermixes [(+) sucrose] and not added in the other half ofthe 5×RT-PCR mastermixes [(−) sucrose]. One set of (+) sucrose and (−)sucrose 5×RT-PCR mastermixes were dried down by lyophilization andincubated at 45° C. The other set of (+) sucrose and (−) sucrose5×RT-PCR mastermixes were dried down at 45° C. in uncapped tubes andincubated at 45° C. Stability of the Dry Reaction 5×RT-PCR mastermixeswas determined by performing RT-PCR over a 3 month period.

Example 2: Analysis of the Dry Reaction Mastermixes by RT-PCR

The Dry Reaction RT-PCR Mastermixes were reconstituted to 1×concentration and analyzed in RT-PCR reaction. Reverse transcription andamplification of the RNA target was performed according to the protocolof the GeneAmp® Gold RNA PCR Reagent Kit (P/N 4308206, AppliedBiosystems, Foster City, Calif.). Briefly, 1×10⁴ input copies of controlpAW109 RNA transcript was added to 0.2 μM each of DM151 and DM152primers which yields a 308 base pair target from the IL-1α gene. Theamplified product was detected using the TaqMan probe AL42F at 0.1 μMconcentration. In some reactions, manganese acetate (Mn(OAc)₂) was addedto a final concentration of 1.5 mM. RT-PCR was performed on aLightCycler® 480 Instrument (Roche Diagnostics) with the reversetranscription phase conducted at 55° C. for 5 minutes, 60° C. for 5minutes and 65° C. for 5 minutes, followed by PCR at 92° C. for 15seconds and 60° C. for 40 seconds with 55 cycles.

Example 3: Results of RT-PCR Reactions

RT-PCR reactions were performed on the Dry Reaction RT-PCR Mastermixeswhich had been stored for 1 day, 15 days, 29 days, 49 days, and 90 daysand the results of the RT-PCR reactions conducted on each of the daysare depicted in the growth curves shown in FIGS. 1-5. After 1 day, boththe lyophilized and the dried down (+) sucrose mastermixes were stablewhile the lyophilized (−) sucrose mastermix was completely degraded andthe dried down (−) sucrose mastermix started to show degradation (FIG.1A, B). After 15 days, both the lyophilized and dried down (+) sucrosemastermixes were stable and showed similar growth curves (FIG. 2A,B).After 29 days, the lyophilized (+) mastermixes started showingdegradation with more degradation observed in mastermixes not containingmanganous acetate (FIG. 3A). In contrast, the dried down (+) mastermixeswere still stable with growth curves that showed little difference fromthe growth curves on day 15 (FIG. 3B). After 49 days, the lyophilized(+) sucrose mastermixes were completely degraded (FIG. 4A) whereas thedried down (+) sucrose mastermixes containing Mn(OAc)₂ was still stable.Finally, after 90 days, the dried down (+) sucrose mastermixes with noMn(OAc)2 was completely degraded and the mastermixes with Mn(OAc)2showed some degradation but were still able to generate growth curves(FIG. 5). These results clearly show not only the stabilizing effects ofsucrose and manganese acetate but also the superior performance of anon-lyophilized mastermix when compared with a lyophilized mastermix.

Example 4: Preparation and Analysis of Dry Reaction 5×RT-PCR

Mastermixes at Ambient TemperatureDry Reaction 5×RT-PCR mastermixes withthe composition as described in Example 1 (with or without sucrose inthe master mix) were pipetted into 2 ml polypropylene tubes. Mn(OAc)₂was added to half of the tubes and the other half were left withoutMn(OAc)₂. These samples where dried down at 45° C. and at atmosphericpressure in the uncapped 2 ml polypropylene tubes. At the end of 7 daysat 45° C., the 2 ml tubes were capped and stored at ambient roomtemperature protected from light. Stability time points were determinedover a one year period. These mastermixes were analyzed by RT-PCRreactions using the amplification reagents and conditions as describedin Example 2.

The results of the RT-PCR reactions are depicted in the growth curvesshown in FIGS. 6-11. FIG. 6 shows that after 26 days at ambient (room)temperature, the dried down RT-PCR mastermixes with sucrose were stableand generated curve growths whereas the dried down RT-PCR mastermixeswithout sucrose were completed degraded and generated no growth curves.The RT-PCR mastermixes with sucrose showed stability in storage atambient temperature after 71 days (FIG. 7), 222 days (FIG. 8), 299 days(FIG. 9), 353 days (FIGS. 10) and 379 days (FIG. 11). RT-PCR mastermixthat was dried down with added Mn(OAc)₂ showed equal or greaterstability than the same material dried down without the metal. TheseRT-PCR results showed that efficient amplification was achieved with theRT-PCR Master Mix formulations with sucrose after more than 12 months ofsitting in the dark at ambient (room) temperature.

What is claimed is:
 1. A dry reaction mixture composition comprising atleast one nucleic acid amplification-related enzyme, nucleotidetriphosphates, manganese acetate (Mn(OAc)₂), and sucrose, wherein thecomposition is air-dried at 45° C. and is non-lyophilized or non-vacuumdried, and wherein the composition has improved stability and performsbetter compared to a reaction mixture which is (i) lyophilized orvacuum-dried; and (ii) lacking sucrose.
 2. The composition of claim 1,wherein the dry reaction mixture retains activity upon storage underconditions that are, or are equivalent to, 45° C. for 3 months.
 3. Thecomposition of claim 1, further comprising at least one componentselected from the group consisting of an aptamer, a detergent, a buffer,a salt, and an oligonucleotide.
 4. The composition of claim 1, whereinsaid at least one nucleic acid amplification-related enzyme is athermostable polymerase selected from the group consisting of Thermusaquaticus Taq DNA polymerase, Thermus sp. Z05 polymerase, Thermus sp.Z05-D polymerase, Thermus flavus polymerase, Thermotoga maritimapolymerase, TMA-25 polymerase, TMA-30 polymerase, Tth DNA polymerase, aswell as modified thermostable polymerases, or any combination thereof.5. The composition of claim 4 wherein said at least one nucleic acidamplification-related enzyme is a thermostable polymerase selected fromThermus sp. Z05 polymerase or Thermus sp. Z05-D polymerase.
 6. Thecomposition of claim 1, wherein sucrose is present in an amount suchthat if the composition is reconstituted in aqueous solution, theconcentration of sucrose is between about 50 mM to about 1000 mM.